Supplementary Materials and Methods

Supplementary Materials and Methods Flies. Flies were raised on a 12 h:12 h light:dark cycle on standard cornmeal-yeast agar medium at 25°C and 60% humidity. aayKG (BDSC #14135), stdhpBac (BDSC #11438), aay RNAi #3 (BDSC #38338), 23E10-Gal4 (BDSC #49032), and ChAT-Gal80 (BDSC #60321) were obtained from the Bloomington Drosophila Stock Center. aayKG and stdhpBac flies were outcrossed six times to iso31 flies (BDSC #5905). aay RNAi #1 (VDRC #110661) was obtained from the Vienna Drosophila Resource Center. aay RNAi #2 (#3705R-3) was obtained from NIG-FLY. elav-Gal4, nrv2-Gal4, and UAS-NaChBac were used previously in the lab. Gal4 lines used for screening with aay RNAi #1 are as follows: OK107 (1) (lab stock), c739 (2) (lab stock), 201y (3) (lab stock), 104y (4) (kindly provided by Dr. J Douglas Armstrong), Dsk (5) (lab stock), Dh44 (6) (kindly provided by Dr. Greg Suh), Ilp2 (7) (lab stock), Lk (8) (lab stock), Pdf (9) (lab stock), Cry (10) (lab stock), tim (11) (lab stock), Tdc2 (7) (lab stock), TH (12) (lab stock), OK371 (13) (lab stock), ChAT-7.4 (Cha) (14) (lab stock), 55D01 (BDSC #39110), and ppk (BDSC #32079). For expression patterns of 55D01-Gal4, please refer to FlyLight expression data (http://flweb.janelia.org/cgibin/view_flew_imagery.cgi?line=R55D01). Brain enrichment analysis of starvation regulated genes. Briefly, we compared averaged RNA-seq reads between control FD6 brain samples (2 replicate) vs. 9 different head samples from the Flybase database (ftp://ftp.flybase.net/releases/FB2017_06/precomputed_files/genes/gene_rpkm_report_fb_20 17_06.tsv.gz). Differential gene expression was assessed by t-test, using the limma package (15). Quantitative PCR. Individual 5–7-day-old male flies, in a 12 h:12 h light:dark cycle, were collected and sacrificed by freezing at -80°C. Heads were removed by vortexing and isolated on frozen sieves. Frozen heads were homogenized, and total RNA was extracted from 20 heads per sample with Trizol (Invitrogen). RNA (1 µg) was used for cDNA synthesis using

M-MLV Reverse Transcriptase (Promega). qPCR was performed using PrimeQ-Mastermix (Genetbio) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The qPCR primers used in this study were as follows: 5’-AGC GAC GAT TCC CTA ATC AC-3’ (forward) and 5’-ACA TTG CCA CCG AAA CCT AT-3’ (reverse) for aay; 5’-CGT CAC ATC CGC TGC CTT TC-3’ (forward) and 5’-TCA CTC CGG TTC GCT TCT GT-3’ (reverse) for stdh; 5’-CAC CAG GAA CTT CTT GAA TCC GG-3’ and 5’-AGA TCG TGA AGA AGC GCA CCA AG-3’ for RpL32 as an internal control. Generation of aay polyclonal antibody. A bacterial expression vector encoding HIS-AAY was transformed into BL21 competent E. coli cells (Enzynomics). Expression of HIS-AAY protein was induced with 0.5 mM Isopropyl-β-D-thiogalactoside (IPTG) overnight at 18°C. Cells were harvested and sonicated using six 10 second bursts. Following sonication, cells were lysed in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Lysates were centrifuged, and the supernatant containing His-tagged AAY was purified using a Ni-NTA resin (Thermo) according to the manufacturer’s instructions and dialyzed against a dialysis buffer (25 mM Tris-Cl, 300 µM NaCl, and 30% glycerol). The purified recombinant protein was injected into two rabbits. Light-induced arousal. Five- to seven-day-old male flies were individually loaded into 65 × 5 mm glass tubes plugged with 5% sucrose/1% agar in PCR tubes and incubated in 12 h:12 h light:dark cycle for three days. On the fourth day, flies were transferred to 5% sucrose or 1% agar on ZT0 (lights on) and a single light pulse was given on ZT18 (3 a.m.) for 20 seconds. Flies that displayed any locomotion during 5 minutes (sleep = greater than or equal to 5 minutes of inactivity) prior to light-pulse were excluded from analysis. For each fly that was sleeping, any movement within 5 minutes of light-pulse (light-pulse bin included) was regarded as a positive response to stimulation. Startle-induced negative geotaxis assay. 13-17 male flies (5-7 days old) raised on standard cornmeal-yeast agar medium were transferred to an empty polystyrene vial apparatus (two polystyrene vials vertically joined by tape, facing each other) without anesthesia. Flies were

allowed to acclimatize to apparatus for 10 minutes before starting assay. For each trial, flies were tapped gently, but rapidly to the bottom of the vial and the number of flies that passed the 8 cm mark (from the bottom of the vial) in 10 seconds were scored. This was repeated ten times with 1 minute rest intervals between each trial. 10 trials constituted 1 experiment and percentage of flies that passed the 8cm mark were average over 5 experiments for each genotype. Sleep rebound. Mechanical sleep deprivation was carried out with Sleep Nullifying Apparatus (SNAP) (16). Baseline sleep was recorded on the SNAP for three days and flies were mechanically sleep deprived for 12 hours, starting from ZT12 (lights off) at day 4 until ZT0 (lights on) at day 5. Sleep rebound was measured for the initial 12 hours (ZT0 – ZT12) at day 5. Cumulative percentage sleep loss was calculated in individual flies and averaged for each genotype. MCA treatment. For MCA treatment, five-to-seven-day-old male flies were monitored in 5% sucrose/1% agar food for 4 days. On the fifth day, PCR tubes with 1% agar supplemented with either 200 µM or 800 µM MCA (Sigma) replaced the previous food at ZT0 and flies were monitored for 24 hours. Percentage sleep change was calculated during the night-time (ZT12 - ZT24) of MCA treatment day, since the majority of the exaggerated starvationinduced sleep suppression in stdh mutants were observed during the night-time. Triglyceride and protein assays. Five- to seven-day-old male flies were placed in 5% sucrose/1% agar food in groups of eight and entrained in a 12 h:12 h light:dark cycle for 3 days. On the fourth day, flies were divided into two groups: the first group was sacrificed at ZT0 (non-starved), and the second group was starved for 18 hours in 1% agar. After starvation, the second group of flies were also sacrificed. Triglyceride and protein measurements were made in whole flies according to the manufacturer’s instructions using the Infinity Triglycerides kit (Thermo) and the BCA Protein Assay kit (Thermo).

Survival under starvation. To measure survival during starvation, five- to seven-day-old male flies were loaded in DAM on 5% sucrose/1% agar food and were incubated for two days for acclimatization. Starvation began on ZT0 of day 3 and locomotor activity was monitored every 60 minutes until death (locomotor count = 0) for each fly. Number of flies that died at each time point were counted manually and a Kaplan-Meier curve was drawn for each genotype. Statistics. All statistical analyses were performed with Prism 7 (GraphPad). Pairwise comparisons were evaluated by two-tailed unpaired t-tests. One-way ANOVA followed by Tukey’s post-hoc test was used when the effect of a single independent variable was assessed (e.g., genotype) among more than two genotypes. Two-way ANOVA followed by Tukey’s post hoc test was used when the interactive effect of two independent variables were assessed (e.g., genotype vs. starvation and aay mutation vs. stdh mutation).

Supplementary References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Connolly JB, et al. (1996) Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies. Science 274(5295):2104-2107. Yang MY, Armstrong JD, Vilinsky I, Strausfeld NJ, & Kaiser K (1995) Subdivision of the Drosophila mushroom bodies by enhancer-trap expression patterns. Neuron 15(1):45-54. Aso Y, et al. (2009) The mushroom body of adult Drosophila characterized by GAL4 drivers. J Neurogenet 23(1-2):156-172. Donlea JM, Thimgan MS, Suzuki Y, Gottschalk L, & Shaw PJ (2011) Inducing sleep by remote control facilitates memory consolidation in Drosophila. Science 332(6037):1571-1576. Park D, Veenstra JA, Park JH, & Taghert PH (2008) Mapping Peptidergic Cells in Drosophila: Where DIMM Fits In. Plos One 3(3). Dus M, et al. (2015) Nutrient Sensor in the Brain Directs the Action of the Brain-Gut Axis in Drosophila. Neuron 87(1):139-151. Crocker A, Shahidullah M, Levitan IB, & Sehgal A (2010) Identification of a Neural Circuit that Underlies the Effects of Octopamine on Sleep:Wake Behavior. Neuron 65(5):670-681. Murakami K, et al. (2016) translin Is Required for Metabolic Regulation of Sleep. Current Biology 26(7):972-980. Renn SCP, Park JH, Rosbash M, Hall JC, & Taghert PH (1999) A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 99(7):791-802.

10. 11.

12. 13. 14. 15. 16.

Klarsfeld A, et al. (2004) Novel features of cryptochrome-mediated photoreception in the brain circadian clock of Drosophila. Journal of Neuroscience 24(6):1468-1477. Kaneko M, Park JH, Cheng YZ, Hardin PE, & Hall JC (2000) Disruption of synaptic transmission or clock-gene-product oscillations in circadian pacemaker cells of Drosophila cause abnormal behavioral rhythms. Journal of Neurobiology 43(3):207233. Friggi-Grelin F, Iche M, & Birman S (2003) Tissue-specific developmental requirements of Drosophila tyrosine hydroxylase isoforms. Genesis 35(3):175-184. Robinson JE, Paluch J, Dickman DK, & Joiner WJ (2016) ADAR-mediated RNA editing suppresses sleep by acting as a brake on glutamatergic synaptic plasticity. Nat Commun 7. Salvaterra PM & Kitamoto T (2001) Drosophila cholinergic neurons and processes visualized with Gal4/UAS-GFP. Brain Res Gene Expr Patterns 1(1):73-82. Ritchie ME, et al. (2015) limma powers differential expression analyses for RNAsequencing and microarray studies. Nucleic Acids Res 43(7):e47. Seugnet L, et al. (2009) Identifying Sleep Regulatory Genes Using a Drosophila Model of Insomnia. Journal of Neuroscience 29(22):7148-7157.

Up-regulated Down-regulated

Brain enrichment (%)

60 50 40 30 20 10 0

FD6

FD24

Supplementary Figure 1. Brain enrichment of genes regulated by short-term and long-term starvation. Genes up-regulated by short-term starvation (FD6) tended to be enriched more in the brain than genes up-regulated by long-term starvation (FD24), whereas the opposite trend was evident in down-regulated genes.

Ctrl FD6

Ctrl FD24

FD6

FD24

Expression level

Log2(Normalized read count)

Supplementary Figure 2. Heat-map of overlapping genes between FD6 and FD24. Heat-map shows the expression level of the overlapping 115 genes in fed (Ctrl FD6 and Ctrl FD24) and starved conditions (FD6 and FD24). Colors indicate the log2 values of normalized read counts for gene expression.

Fed

Relative expression (aay)

3 2.5

**

*

Starved

***

2 1.5 1 ns

0.5 0

iso31

aayKG

Supplementary Figure 3. aay mRNA quantification in head extracts of iso31 control and aay mutants. aay mRNA expression was induced upon starvation in iso31 control flies, while the KG05974 insertion reduced aay expression (n=3). Starvation*genotype [F(1,8)=10.55; P=0.0117]. Two-way ANOVA followed by Tukey post-hoc analysis was used for comparison. ns, P>0.05; *P